The present invention concerns embodiments related to bladder disorder treatment and other embodiments related to cancer treatment.
Bladder Disorders
Approximately one million people in the United States suffer from the bladder disorder interstitial cystitis, which is a chronic painful urinary bladder condition characterized by thinning or ulceration of the bladder epithelial lining (Curhan et al., 1999).
Cystoscopic abnormalities seen in the bladder of patients with this disorder include petechial hemorrhages called “glomerulations” and ulcers that extend into the lamina propria (Hunner's ulcers) (Johansson and Fall, 1990; Skoluda et al., 1974). The most consistent histologic abnormalities include denudation or thinning of the bladder epithelium to 1-2 cell layers (Johansson and Fall, 1990; Skoluda et al., 1974; Tomaszewski et al., 2001). These findings suggest that interstitial cystitis may be caused by an inhibition of normal bladder epithelial cell proliferation, resulting in a loss of epithelial barrier integrity with subsequent exposure of sensory nerve cells in the bladder wall to urinary constituents. However, the pathogenesis of interstitial cystitis is heretofore unknown.
The isolation of an antiproliferative factor (“APF”) peptide that is made uniquely by bladder epithelial cells from interstitial cystitis patients (Keay et al., 2001; Keay et al., 2000) and profoundly inhibits normal bladder epithelial cell growth (Keay et al., 2003) was previously described. U.S. Pat. No. 5,962,645, incorporated by reference herein in its entirety, teaches a purified human antiproliferative factor (APF) isolated from the urine of patients with interstitial cystitis wherein the APF is characterized by a molecular weight of about 1.7 kDa determined by mass spectrometry on a sample in an aqueous acetonitrile solution and a pI range of about 1.38-3.5, and the APF is capable of inhibiting normal human bladder epithelial (HBE) and bladder carcinoma cell proliferation. Picomolar quantities of HPLC-purified APF were able to induce several changes in normal bladder epithelial cells in vitro, including significantly decreased rates of proliferation (Keay et al., 2003) and decreased production of a growth factor required for log-phase growth of bladder epithelial cells (heparin-binding epidermal growth factor-like growth factor, or HB-EGF) (Keay et al., 2000; Keay et al., 2003).
HB-EGF has been previously described (for example, see U.S. Pat. No. 5,811,393). U.S. Pat. No. 6,156,522 describes a method for diagnosing interstitial cystitis in a subject suffering from bladder dysfunction, said method comprising the steps of (a) measuring the levels of HB-EGF-like growth factor in the urine sample of the subject; and (b) comparing said level with normal levels, wherein decreased levels of heparin-binding epidermal growth factor-like growth factor, as compared to levels of heparin-binding epidermal growth factor-like growth factor in a normal population, indicates the presence of interstitial cystitis. U.S. Pat. No. 6,232,289 teaches a method for enhancing bladder epithelial cell proliferation in a subject in need thereof, said method comprising administering to the subject HB-EGF, in an amount effective to enhance bladder epithelial cell proliferation. U.S. Pat. No. 6,376,197 teaches a method for diagnosing a condition such as interstitial cystitis associated with inhibited bladder epithelial cell proliferation comprising the steps of determining the level of epidermal growth factor in urine from the subject; and comparing said level with normal level, according to the following criterion: increased level of epidermal growth factor, as compared to level of epidermal growth factor in a normal population, indicates the presence of the condition.
Microarray analysis indicated that APF can also induce changes in the pattern of cellular gene expression toward a more differentiated phenotype (Keay et al., 2003). Identification of this factor is therefore important for determining its potential role in the pathogenesis of interstitial cystitis and establishing its utility as a biomarker for this disease.
Preliminary characterization of APF indicated that it was a low molecular weight, relatively heat stable peptide (Keay et al., 2000). APF is found in minute quantities in both patient urine specimens and explanted patient bladder cell supernatants, making conventional methods of structural analysis, such as NMR spectroscopy, unfeasible. As described herein, the complete characterization of this potent growth inhibitor provides a novel structure not previously disclosed. The complete characterization was made using a combination of techniques including mass spectrometry, lectin affinity chromatography, enzymatic analysis, and total synthesis.
Confirmation of the structure of APF was provided using microcapillary liquid chromatography of native and synthetic APF derivatives, as well as by demonstration of synthetic APF's ability to regulate growth factor production and bladder epithelial cell proliferation. Additional evidence of APF's identity was provided by identification of mRNA that bound to a probe for the frizzled 8-protein segment in cells from interstitial cystitis patients but not controls, as well as by binding of rabbit antibodies raised against synthetic APF to purified native APF from supernatants of bladder epithelial cells of interstitial cystitis patients.
Another factor considered to be a cell cycling inhibitory factor is described in U.S. Pat. No. 5,916,871, wherein the factor comprises a sialylated glycopeptide preferably having a molecular weight much larger (e.g. 18 kD or 66 kD) than APF and that inhibits the G1 phase (but not G2) of the cell cycle. This previously described factor also had a carbohydrate component that accounted for less than 10% of its total mass, whereas APF in its native form has carbohydrate that accounts for 44% of its total mass.
Cancer
Cancer continues to be a significant health problem worldwide, and therapies for cancer are in demand. A therapeutic and/or preventative regimen for cancer could include a natural antiproliferative factor, or synthetic analog thereof. The naturally-occurring antiproliferative factor is present in individuals with interstitial cystitis (IC), a devastating disease of the urinary bladder that is characterized by thinning or even focal obliteration of the bladder epithelium. In fact, urine from IC patients has been shown to contain an antiproliferative factor (APF) that decreases 3H-thymidine incorporation by human bladder epithelial cells (Keay et al., 1996). A variety of techniques including total synthesis were previously used to identify APF as a nonapeptide (TVPAAVVVA; SEQ ID NO:1) containing a 2,3-sialylated core 1α-O-linked disaccharide (Galβ1-3GalNAc, the Thomsen-Friedenreich antigen, or “TFag”) linked to the N-terminal threonine residue (i.e., Neu5Acα2-3Galβ1-3GalNAcα-O-TVPAAVVVA; SEQ ID NO:1) (Keay et al., 2004). The peptide sequence of APF is identical to a segment of the 6th transmembrane domain of the frizzled-8 protein, a Wnt ligand receptor (Keay et al., 2004; Saitoh et al., 2001).
Early studies indicated that purified native APF increased E-cadherin expression and decreased proliferation of bladder epithelial cells in vitro (Keay et al., 2003), and both native and synthetic APF were shown to inhibit the proliferation of normal bladder epithelial as well as cells derived from urothelial carcinomas at picomolar to low nanomolar concentrations (Keay et al., 2004; Keay et al., 2006). Therefore, APF, and other derivatives, including more efficacious synthetic derivatives, represent an innovative group of anti-tumor agents with a novel mode of action.
Other and further objects, features, and advantages will be apparent from the following description of the presently preferred embodiments of the invention, which are given for the purpose of disclosure.